The invention relates to the use of inhibitors of calcineurin to prevent glutamate neurotoxicity.
Nitric oxide (NO) has been demonstrated to mediate neuronal relaxation of intestines (Bult (1990) Nature, 345:346-347; Gillespie (1989) Br. J. Pharmacol., 98:1080-1082; and Ramagopal (1989) Eur. J. Pharmacol., 174:297-299) and to mediate stimulation by glutamate of cGMP formation (Bredt (1989) Proc. Natl. Acad. Sci. USA 86:9030-9033). Glutamate, the major excitatory neurotransmitter in the brain, acts through several receptor subtypes, some of which stimulate the formation of cGMP (Ferrendelli (1974) J. Neurochen. 22:535-540). Glutamate, acting at N-methyl-D-aspartate (NMDA) subtype of receptors, is responsible for neurotoxic damage in vascular strokes. Glutamate neurotoxicity has also been implicated in neurodegenerative disorders such as Alzheimer""s and Huntington""s diseases (Choi (1990) J. Neurosci. 10:2493-2501; and Meldrum (1990) Trends in Pharmiacol. Sci. 11:379-387). Selective antagonists of NMDA glutamate receptors prevent neuronal cell death in animal models of hypoxic-ischemic brain injury (Choi (1990) J. Neurosci., 10:2493-2501). In addition, inhibitors of nitric oxide synthase prevent neuronal cell death (Dawson, Proc. Natl. Acad. Sci., USA, 88:6368 (1991)).
Besides their roles in the immune system, the immunophilins, cyclophilin and FK-506 binding protein (FKBP), are highly concentrated in the brain in discrete neuronal structures where they are co-localized with the Ca+2 activated phosphatase, calcineurin (Steiner, et al., Nature, 358:584-587 (1992). Liu (Cell, 66:807-815 (1991)) demonstrated that very low concentrations of FK-506 and cyclosporin A, which bind to FKBP and cyclophilin, respectively, inhibit calcineurin, and Steiner showed that both drugs enhance the phosphorylation of a number of proteins in the brain. Glutamate neurotoxicity acting via N-methyl-D-aspartate (NMDA) receptors is implicated in neuronal damage associated with strokes and neurodegenerative diseases (Choi, Neuron, 1:623-634 (1988); Meldrum, et al., Trends Pharmacol. Sci., 11:379-387 (1990); Choi, Science, 258:241-243 (1992)). In primary cerebral cortical cultures (Dawson, et al., Proc. Natl. Acad. Sci., USA, 88:6368-6371 (1991)), hippocampal slices (Izumi, et al., Neurosci. Lett., 135:227-230 (1993)), and in animal models of focal ischemia Nowicki, et al., Euro. J. Pharma., 204:339-340 (1991)), NMDA toxicity is mediated, at least in part, by nitric oxide (NO), as NO synthase (NOS) inhibitors block this toxicity.
Effective methods of preventing, treating or ameliorating diseases caused by glutamate neurotoxicity are needed in the art.
It is an object of the invention to provide a method of treating diseases caused by glutamate neurotoxicity.
It is another object of the invention to provide a method of treating vascular stroke and neurodegenerative diseases.
It is another object of the invention to provide a method of screening compounds to identify neuroprotective drugs.
These and other objects of the invention are provided by one or more of the embodiments described below. In one embodiment of the invention, a method for treating vascular stroke and neurodegenerative disease patients to block glutamate-mediated neurotoxicity is provided. The method comprises: administering to a vascular stroke or neurodegenerative disease patient a drug which binds to an immunophilin, in an amount effective to inhibit glutamate-mediated neurotoxicity.
In another embodiment of the invention, a method is provided for treating vascular stroke and neurodegenerative disease patients to block glutamate-mediated neurotoxicity. The method comprises: administering to a vascular stroke or neurodegenerative disease patient a drug which binds to an immunophilin, in an amount effective to inhibit calcineurin.
In still another embodiment of the invention, a method is provided for screening compounds to identify neuroprotective drugs. The method comprises the steps of: applying an immunophilin-binding test compound to cultured mammalian neuronal cells; applying a neurotoxic agent selected from the group consisting of NMDA and glutamate to said cultured mammalian neuronal cells; assessing toxicity by determining viability of the cultured mammalian neuronal cells, a neuroprotective drug being identified when a test compound inhibits the toxic effects of said neurotoxic agent.
Thus the present invention provides the art with methods for treating neurological diseases associated with glutamate neurotoxicity, as well as methods of identifying other pharmacological agents which will block glutamate neurotoxicity.